Inhibin secretion in the mare: localization of inhibin alpha, betaA, and betaB subunits in the ovary

To determine the source of circulating inhibin and estradiol-17beta during the estrous cycle in mares, the cellular localization of the inhibin alpha, betaA, and betaB subunits and aromatase in the ovary was determined by immunohistochemistry. Concentrations of immunoreactive (ir-) inhibin, estradiol-17beta, progesterone, LH, and FSH in peripheral blood were also measured during the estrous cycle in mares. Immunohistochemically, inhibin alpha subunits were localized in the granulosa cells of small and large follicles and in the theca interna cells of large follicles, whereas inhibin betaA and betaB subunits were localized in the granulosa cells and in the theca interna cells of large follicles. On the other hand, aromatase was restricted to only the granulosa cells of large follicles. Plasma ir-inhibin concentrations began to increase 9 days before ovulation; they remained high until 2 days before ovulation, after which they decreased when the LH surge was initiated. Thereafter, a further sharp rise in circulating ir-inhibin concentrations occurred during the process of ovulation, followed by a second abrupt decline. After the decline, plasma concentrations of ir-inhibin remained low during the luteal phase. Plasma estradiol-17beta concentrations followed a profile similar to that of ir-inhibin, except during ovulation, and these two hormones were positively correlated throughout the estrous cycle. Plasma FSH concentrations were inversely related to ir-inhibin and estradiol-17beta. These findings suggest that the dimeric inhibin is mainly secreted by the granulosa cells and the theca cells of large follicles; granulosa cells of small follicles may secrete inhibin alpha subunit, and estradiol-17beta is secreted by the granulosa cells of only large follicles in mares.     

Pituitary activin receptor subtypes and follistatin gene expression in female rats: differential regulation by activin and follistatin

The activins, hormones produced in the gonads and extragonadal tissues (including the pituitary), rapidly increase FSH beta messenger RNA (mRNA) and FSH secretion. In the rat, activin acts via a family of activin receptor (ActR) subunits that includes at least one type I (ActRI or ALK-2) and two homologous type II (IIA and IIB) subunits. We have previously reported that ActRIIA mRNA rises after ovariectomy (OVX). Potentially, the OVX-induced increases in ActR mRNAs could result from altered activin or the activin-binding protein follistatin. It was the purpose of the current studies to determine whether activin and/or follistatin regulated activin receptor subunit mRNAs. Adult female rat pituitaries were dissociated and plated for 48 h, transferred to wells containing follistatin or activin for 2 or 24 h, then RNA extracted for measurement of ActRI, IIA, and IIB and follistatin mRNAs. All three ActR mRNAs were easily detectable in pituitary RNA, with the relative abundance of ActRI > IIA > IIB (18:9:1). Between 2-24 h, levels of all three ActR mRNAs increased 2- to 3-fold in wells containing medium alone, whereas levels of follistatin mRNA were unchanged. Follistatin significantly reduced FSH secretion and follistatin mRNA, but not the ActR mRNAs. Activin increased ActRI (4-fold, at 2 h), ActRIIB (2-fold, at 24 h), and follistatin (2-fold, at 24 h) mRNAs and FSH release (2-fold, at 24 h), but did not alter ActRIIA mRNA levels. We conclude that 1) pituitary ActR mRNA expression is under inhibitory tone in vivo, as suggested by the effect of pituitary removal and cell dispersion and an earlier report after OVX. 2) Pituitary-derived activin stimulates follistatin (but not ActR) mRNA production, and additional increases in follistatin mRNA can be induced by exogenous activin. 3) Higher concentrations of activin differentially regulate pituitary ActR mRNA expression, suggesting that activin exerts a positive feedback effect on its own receptor.     

Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels

To elucidate the role of germ cells in the regulation of inhibin B secretion, serum inhibin B levels in prepubertal boys and adult men whom had a concurrent testicular biopsy showing either normal or impaired testicular function were compared. In addition, by immunohistochemistry the cellular localization of the two subunits of inhibin B (alpha and betaB) were examined in adult testicular tissue with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only tubules (SCO) as well as in normal testicular tissue from an infant and a prepubertal boy. Adult men with testicular biopsy showing normal spermatogenesis (n=8) or spermatogenic arrest (n=5) had median inhibin B levels of 148 pg/mL (range, 37-463 pg/mL) and 68 pg/mL (range, 29-186 pg/mL), respectively, corresponding to normal or near-normal levels of our reference population (165 and 31-443 pg/mL; n=358). Men with SCO (n=9) had undetectable or barely detectable (n=1) serum levels of inhibin B. In contrast to adults, prepubertal boys with SCO (n=12) all had measurable serum inhibin B levels that corresponded to our previously determined normal range in healthy prepubertal boys (n=114). However, in postpubertal samples from the same SCO boys, inhibin B levels were undetectable as in the adult SCO men. Intense inhibin alpha-subunit immunostaining was evident in Sertoli cells in both prepubertal and adult testes. In the prepubertal testis, positive immunostaining for the betaB-subunit was observed in Sertoli cells. In the adult testis, intense immunostaining for the betaB-subunit was evident in germ cells from the pachytene spermatocyte to early spermatid stages and to a lesser degree in Leydig cells, but not in Sertoli cells or other stages of germ cells. Thus, surprisingly, in adult men the two subunits constituting inhibin B were expressed by different cell types. We speculate that during puberty Sertoli cell maturation induces a change in inhibin subunit expression. Thus, immature Sertoli cells express both alpha and betaB inhibin subunits, whereas fully differentiated Sertoli cells only express the alpha-subunit. The correlation in adult men between serum inhibin B levels and spermatogenesis may be due to the fact that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells, including the stages from pachytene spermatocytes to early spermatids.     

Intrathecal chemotherapy for treatment of overt meningeal leukemia: comparison between intraventricular and traditional intralumbar route

The developmental changes in the expression of mRNAs encoding the alpha 1 and alpha 2 subunits of inhibitory glycine receptors in the spinal cord of fetal and postnatal rats were examined by in situ hybridization. During embryonic periods (E11-18), the mantle zone was scarce in the alpha 1 mRNA, but the germinal zone (matrix layer) at E11-14 expressed higher levels of the message. At postnatal day 0 (P0), the alpha 1 signals became manifested throughout the gray matter of the spinal cord. The intensities of the signals were increased to reach a maximal level at P21. By contrast, the spinal tissues at P0 exhibited the highest levels of alpha 2 mRNA, which decreased with the postnatal development. In P50 rats, the alpha 2 mRNA was barely expressed in the ventral horn, but a significant number of grains could still be detectable in a population of cells in the dorsal horn. During postnatal development from P0 to P10, the spinal tissues were rich in the alpha 1 and alpha 2 mRNAs, both of which were detected in the presumed motoneurons. The coexistence of the two subunits in single neurons might correlate with the modification of the glycine receptor function during the development of the spinal cord.     

Expression of messenger ribonucleic acid for inhibin subunits and ovarian secretion of inhibin and estradiol at various stages of the sheep estrous cycle

The relationship between expression of inhibin mRNA and ovarian secretion of estradiol (E2) and immunoactive inhibin was investigated at midluteal phase and throughout the follicular phase of the sheep estrous cycle. At laparotomy, timed samples of ovarian blood were collected and ovaries were removed from 39 Scottish Blackface ewes (ovulation rate 1.3 +/- 0.1) on Day 10 of the luteal phase or 24, 48, 60, 72, or 84 h after injection of cloprostenol (PG; 100 micrograms) on Days 10-12. Ovaries were removed and fixed for in situ hybridization using 35S-labeled antisense riboprobes transcribed from inhibin alpha, beta A, and beta B cDNAs. LH, E2, and inhibin concentrations were determined by RIA. On the basis of peripheral LH levels and the presence of estrogen-active follicles (E-A; > or = 3 mm in diameter secreting > 1 ng/min E2) or recent ovulations, animals were grouped as follows: presurge (24 or 48 h post-PG; LH < 5 ng/ml; n = 7), midsurge (with E-A; LH > 5 ng/ml; n = 6), late surge (large follicle not E-A; LH > 5 ng/ml; n = 4), postsurge (large follicle not E-A; LH < 5 ng/ml; n = 7), and postovulation (n = 10). As expected, E2 secretion by the "active" ovary (containing preovulatory follicle) tended to increase with follicular development such that secretion was maximal at midsurge and then declined. E2 secretion by the "inactive" ovary was low at all stages. Immunoactive inhibin, in contrast, was secreted in substantial quantities by both ovaries, although secretion from active ovaries was higher at all stages (p < 0.05). Effects of stage on secretion were not significant, but immunoactive inhibin secretion from active ovaries was high in postsurge animals when E2 secretion was very low. Hybridization for inhibin mRNA was specific for granulosa cells of antral follicles. While most sheep in the luteal (4 of 5), presurge (2 of 3), and midsurge groups (5 of 5) had at least one inhibin-positive large follicle (expressing both alpha- and beta-subunit mRNA), none were present between the LH surge and ovulation (late and postsurge groups). Inhibin mRNA was undetectable in midcycle CL, but 4 of 10 recent ovulations hybridized weakly with the alpha probe and one very weakly with the beta A probe. The mean number of inhibin-positive large follicles per animal (in those having at least one) was 1.3 +/- 0.15 (n = 15 ewes).(ABSTRACT TRUNCATED AT 400 WORDS)     

Exposure to prenatal nicotine transiently increases neuronal nicotinic receptor subunit alpha7, alpha4 and beta2 messenger RNAs in the postnatal rat brain

This study determined the effects of prenatal nicotine exposure (2 mg/kg/day) in Sprague Dawley CD rats via subcutaneously implanted osmotic minipumps, during gestational days 7-21, on postnatal levels of neuronal nicotinic receptor alpha4, alpha7 and beta2 subunit messenger RNAs. Northern analysis of postnatal day 1, 7, 14 and 28 hippocampal/septal and cortical total RNA using alpha-[32P]dCTP-labeled alpha4, alpha7 and beta2 complementary DNA probes identified a single (5.7-kb) alpha7 messenger RNA, three (2.4-, 3.8- and 8.0-kb) alpha4 messenger RNAs and four (3.7-, 5.0-, 7.5- and 10.0-kb) beta2 messenger RNAs. In comparison to prenatal saline, prenatal nicotine produced several significantly higher messenger RNA levels (cortical: 5.7-kb alpha7, 2.4-, 3.8- and 8.0-kb alpha4, 10.0-kb beta2; hippocampal/septal: 2.4- and 8.0-kb alpha4); these increases occurred predominantly on, but were not restricted to, postnatal day 14. Effects of nicotine were generally resolved by postnatal day 28. Collapsing the data across sex and age, a significant treatment effect indicated that hippocampal/septal and cortical 8.0-kb alpha4 messenger RNA levels and 10.0-kb beta2 messenger RNA levels were significantly higher following prenatal nicotine exposure. This is the first study indicating that prenatal nicotine produces alterations in developing postnatal rat neuronal nicotinic receptor messenger RNA levels, possibly by premature stimulation of neuronal nicotinic receptors. These results further implicate the teratogenic potential of nicotine in postnatal neuronal development.     

Expression of inhibin alpha and inhibin/activin beta A subunits in the granulosa layer of the large preovulatory follicles of the hen

The expression of the mRNA for the inhibin/activin subunits (alpha and beta A) in the granulosa layer of the five largest preovulatory follicles of the hen was investigated. Total RNA from the granulosa layer of the F5 (the fifth largest) to F1 (the largest) follicles was extracted and analyzed by Northern blot analysis using homologous chicken inhibin alpha and beta A subunit cDNA probes. RNA loading was quantified by a cDNA probe of bovine 18S rRNA. Results showed that for the chicken inhibin alpha subunit mRNA signals (n = 3), the mean relative intensity for the F1, F2, F3, and F4 follicles was 0.50 +/- 0.10 ( +/- SEM,), 0.52 +/- 0.08, 0.59 +/- 0.06, and 0.81 +/- 0.04, respectively, compared to a mean relative intensity of 1.00 (p < 0.05) for the F5 follicle. For the beta A subunit mRNA signals (n = 3), the mean relative intensity for the F5, F4, F3, and F2 follicles was 0.25 +/- 0.06, 0.28 +/- 0.15, 0.40 +/- 0.17, and 0.48 +/- 0.10 (p < 0.05) for the F1 follicle. The inhibin alpha subunit was also estimated to be more abundantly expressed among follicles in the granulosa layer than was the beta A subunit. Our data indicate that the expression of inhibin alpha and beta A subunits is differentially regulated in the hen granulosa layer during follicular development. Expression of the alpha subunit is reduced with follicular development whereas inhibin beta A subunit expression is dramatically enhanced. In addition, the granulosa layer of the large preovulatory follicles may produce more inhibin alpha subunit than beta A subunit, and the F1 follicle may be the primary source of the beta A subunit for dimeric inhibin and/or activin in the hen.     

Staff attitudes that impede the implementation of behavioral treatment programs

The formation of cardiac cushion tissue, which ultimately contributes to formation of the valves and septa, is dependent on the regional activation of cardiac endothelial cells to undergo an epithelial-mesenchymal transition. This endothelial transition was correlated with activin betaA mRNA expression by Northern and in situ hybridization in both a temporal and spatial manner in developing mouse embryos. Activin betaA was the only subunit of the inhibin family detected during the initial phase of endothelial cell transition; activin betaB was detected at later stages, and inhibin alpha was not detectable in the heart. An in vitro assay that has been used to study mesenchymal cell formation in chick was modified for use with mammalian embryos. Conditioned media from embryonic mouse cardiocyte cultures was shown to substitute for the endogenous inductive signal in these assays. The presence of activin betaA was demonstrated by Western blot analysis of the cardiocyte conditioned media (CCM). Modified antisense oligonucleotides to activin betaA inhibited the endothelial-mesenchymal transition in the assay system, which was not affected by control oligonucleotides. Adapting the avian culture system for use with mice enabled the use of tissue from mice with a null allele for activin betaA. CCM produced from embryos homozygous for the mutant betaA allele did not contain activin betaA and was used in in vitro assays. CCM lacking activin betaA produced fewer mesenchymal cells from cardiac endothelial monolayers than CCM with activin betaA. Localized expression of activin betaA in the embryonic heart indicates a possible role in the endothelial-mesenchymal transition. Bioassays in which activin betaA expression is blocked or activin betaA is absent from the media indicate that activin betaA promotes the formation of mesenchymal cells in the endothelial cushions, which are required for normal septation.     

Microtubule-associated protein-2 in the rat testis: a novel site of expression

The testis is one of the most abundant sources of microtubule networks. These networks include mitotic and meiotic spindles, the spermatid manchette and axoneme, and the Sertoli cell cytoskeleton. Microtubules are composed of alpha- and beta-tubulin subunits that are polymerized and stabilized by a variety of microtubule-associated proteins (MAPs). One of these, MAP2, has been extensively characterized as a brain-specific protein with the capacity to bind tubulin, cAMP-dependent kinase, and calmodulin. MAP2 mRNA is processed into at least two variants encoding proteins designated MAP2a, MAP2b, and MAP2c. Of the 5.7 kb of coding sequence in the 9-kb mRNA that encodes MAP2a and MAP2b, a deletion of approximately 4 kb produces mRNA encoding MAP2c, which consists of only the N- and C- terminal regions of MAP2b. To determine whether MAP2 was present in the rat testis, microtubule preparations were isolated from adult rat testis and brain by means of taxol-mediated polymerization and analyzed by gel filtration, ELISA, and Western blotting using polyclonal and monoclonal antibodies reactive with MAP2. A 74-kDa protein corresponding to MAP2c was detected in the testis. These results were confirmed by Northern blot analysis of total RNA from adult rat brain and testis with cDNA probes that distinguish between the known MAP2 splice variants. The predominant mRNAs in testis of 6 kb and 2.5-3.5 kb corresponded to MAP2c. A single 6-kb mRNA with the potential to encode MAP2c was detected in enriched preparations of immature Sertoli cells and adult Leydig cells. Round spermatids contained at least two MAP2 mRNAs between approximately 2.5 and 3.5 kb in size that displayed a stage-specific pattern of expression. Immunohistochemistry showed a MAP2-like protein in both somatic and germ cells, with a particularly distinct localization within the cytoplasm of primary and secondary spermatocytes at stage XIV of the seminiferous cycle during meiotic metaphase. In addition to cytoplasmic staining, a novel localization of this protein was observed in the nucleus of many testicular cells.     

Gonadotropin-releasing hormone regulation of gonadotropin subunit gene expression in female rats: actions on follicle-stimulating hormone beta messenger ribonucleic acid (mRNA) involve differential expression of pituitary activin (beta-B) and follistatin mRNAs

GnRH is the primary stimulus in the regulation of gonadotropin subunit mRNA expression. Additionally, local (pituitary) production of activin and follistatin appear to modulate the expression of FSH beta mRNA. The current studies aimed to determine whether GnRH regulation of pituitary activin (beta-B) and follistatin mRNAs could play a role in the differential actions of GnRH pulse pattern on gonadotropin mRNA expression in female rats. In response to altered GnRH pulse amplitude, the expression of FSH beta and follistatin mRNAs followed an inverse pattern. Only high dose GnRH increased expression of follistatin whereas, in contrast, beta-B and FSH beta expression were increased following lower doses of GnRH. To determine whether increased follistatin mRNA expression was correlated with FSH beta mRNA responses, we examined their temporal relationship following high dose GnRH. Both FSH beta and follistatin mRNAs were increased within 2 h and remained increased through 6 h. However, by 12 h FSH beta mRNA levels returned to values seen in controls, suggesting that increased follistatin requires 6-12 h to reduce FSH beta mRNA. In response to altered GnRH pulse frequency, FSH beta expression was increased at all pulse intervals (8-240 min) examined. Rapid GnRH pulse frequencies (8-min intervals) increased follistatin expression, whereas beta-B mRNA was only increased after 30-min pulse intervals, which also resulted in maximal FSH beta mRNA concentrations. These results suggest that changes in pituitary activin (beta-B) and follistatin mRNA expression may be important components of gonadotrope responses to pulsatile GnRH, and potentially imply that GnRH stimulation of activin and follistatin peptide production provides regulatory control over the production of FSH.     

Extraembryonic expression of the human MHC class I gene HLA-G in transgenic mice. Evidence for a positive regulatory region located 1 kilobase 5' to the start site of transcription

Trophoblast, the only fetal tissue in direct contact with maternal cells, fails to express the polymorphic HLA class I molecules HLA-A and -B, but does express the nonpolymorphic class I molecule HLA-G. It is thought that HLA-G may provide some of the functions of a class I molecule without stimulating maternal immune rejection of the fetal semiallograft. As a first step in identifying the cis-acting DNA regulatory elements involved in the control of class I expression by extraembryonic tissue, several types of transgenic mice were produced. Two HLA-G genomic fragments were used, 5.7 and 6.0 kb in length. These included the entire HLA-G coding region, 1 kb of 3' flanking sequence, and 1.2 or 1.4 kb of 5' flanking sequence, respectively. A hybrid transgene, HLA-A2/G, was produced by replacing the 5' flanking sequence, first exon, and early first intron of HLA-G with the corresponding elements of HLA-A. Comparison of transgene mRNA expression patterns seen in HLA-A2/G and HLA-G transgenic mice suggests that 5' flanking sequences are largely responsible for the differing patterns of expression typical of the classical class I and HLA-G genes. Studies comparing the extraembryonic HLA-G expression levels of founder embryos transgenic for either the 5.7- or 6.0-kb HLA-G transgene showed that the 6.0-kb transgene directed HLA-G expression far more efficiently than did the 5.7-kb HLA-G transgene, producing extraembryonic HLA-G mRNA levels similar to those seen in human extraembryonic tissues. The results of these studies suggest that the 250-bp fragment present at the extreme 5' end of the 6.0-kb HLA-G transgene and absent from the 5.7-kb HLA-G transgene contains an important positive regulatory element. This 250-bp fragment lies further upstream than any of the previously documented class I regulatory regions and may function as a locus control region.     

Ovarian activin receptor subtype and follistatin gene expression in rats: reciprocal regulation by gonadotropins

The production of activin, follistatin (FS), and inhibin, proteins present in the ovary and involved in mammalian reproduction, is regulated by gonadotropins and estradiol. We report here gonadotropin regulation of ovarian activin receptor (ActR) subtype and FS mRNAs. Expression of ActRI, ActRIIA, ActRIIB, and FS mRNA was measured on the afternoon of proestrus (1800 h) and the morning of estrus (0800 h). ActRI and ActIIA subtype mRNA concentrations fell by approximately 50% (p < 0.05) following the proestrous gonadotropin surge (ActRIIB mRNA was undetectable), while FS mRNA was unchanged. To define the contribution of gonadotropins, hypophysectomized (HYPOX) female rats were given recombinant human (rh) FSH and hCG, which decreased both ActR mRNAs (by approximately 70% and aproximately 50% for ActRI and IIA, respectively) and increased FS mRNA by 2-fold. As gonadotropins could act via estradiol (E2), HYPOX rats were given E2; ActRI was decreased, but ActRIIA mRNA was increased. The actions of gonadotropins were preferential, as the combination of rhFSH and hCG with E2 reduced ActRIIA mRNA. FS mRNA was increased to a similar degree by E2 and/or gonadotropins. These data suggest that gonadotropins regulate ActR and FS gene expression via multiple mechanisms. Both a direct action on ActRIIA (inhibition) and an indirect action through E2 on ActRI (inhibition) and FS (stimulation) suggest potential physiologic mechanisms for the reciprocal regulation of ActR subtype and FS mRNAs.     

Presence of activin, inhibin, and follistatin in epithelial ovarian carcinoma

Activin induces proliferation in epithelial ovarian carcinoma cell lines, whereas follistatin (FS), an activin binding protein, inhibits this action. To test the hypothesis that activin production, in excess of inhibin and FS, results in cell proliferation in epithelial ovarian tumors, messenger RNA (mRNA) expression of the activin family of proteins, FS, and activin type I and II receptors was examined in 25 primary epithelial ovarian tumors and tumor epithelium in culture (n = 7) using RT-PCR. Activin A was measured in the serum of ovarian cancer patients, and activin A, total inhibin, and FS protein secretion was measured from primary epithelial tumors in vitro. The effect of activin and FS on cell proliferation was assessed by measuring [3H]thymidine incorporation. All results were compared with normal ovarian epithelium. All epithelial ovarian tumors expressed mRNA for the alpha, beta A, and beta B subunits; FS 288 and 315; and the activin type IA, IB, II, and IIB receptors. beta A mRNA expression, as assessed using semiquantitative RT-PCR, was 3-fold greater in cultured tumor epithelium than in primary tumors (band density 0.86 +/- 0.17 vs. 0.28 +/- 0.09; P < 0.01). In addition, beta A mRNA was abundantly expressed in normal epithelium in culture (n = 2), whereas only trace amounts were seen in 2/9 primary epithelial samples. Activin protein was secreted by 24/25 primary epithelial ovarian tumors (range 0.2-155.8 ng/mL). In contrast, total inhibin was secreted by only 2/25 (range 0.01-0.92 ng/mL), whereas free FS was not detectable in the medium of any tumor (< 0.5 ng/mL). Treatment with activin or FS did not consistently affect cell growth. Measurement of serum activin A in a subset of subjects and in 27 additional subjects with epithelial ovarian carcinoma (n = 33) revealed preoperative activin A levels > 3 SD above the mean for pre- and postmenopausal women in 13/33 (39%) subjects. We conclude that in epithelial ovarian cancer: 1) beta A subunit mRNA is expressed, 2) activin protein is secreted more frequently than inhibin and in greater quantities than FS, 3) beta A subunit mRNA expression is greater in neoplastic and normal epithelium in culture than in the primary tissue, 4) the majority of tumors in culture do not respond to activin or FS treatment with proliferation, and 5) serum activin levels may reflect tumor secretion in some patients. Thus, activin A appears to be available as an autocrine/paracrine factor in epithelial ovarian tumors and may contribute to circulating levels, but its role in tumorigenesis has yet to be defined.     

Direct effects of estradiol and tamoxifen on gene expressions of inhibit alpha- and beta A-subunits in rat granulosa cells in vitro

To analyze direct effects of estrogen on gene expressions of inhibin subunits in vitro, the mRNA levels of inhibin alpha- and beta A-subunits were measured in rat granulosa cells cultured with FSH or estradiol. Through the culture process of the granulosa cells with serum-free medium, both the alpha- and beta A subunit mRNAs decreased, and were partially increased again by adding FSH to the culture medium. To examine whether these FSH effects are mediated via estrogen production, estradiol or tamoxifen was added to the cultured granulosa cells. After 36-h culture with estradiol, the inhibin alpha-subunit decreased but the inhibin beta A-subunit increased, in a dose-responsive manner. Tamoxifen showed completely opposite effects to estradiol, and a combination of estradiol and tamoxifen resulted in similar levels of inhibin-alpha and -beta A mRNAs to the control. These results indicate that estrogen would by a certain pathway affect gene expressions of the inhibin subunits in the rat granulosa cells, and may regulate the production of inhibin and activin through the paracrine system.     

Immunolocalization of inhibin and activin subunits in human endometrium across the menstrual cycle

Inhibin/activin alphaC/alphaN and betaA subunits were localized immunohistochemically in the human endometrium throughout the menstrual cycle using an affinity-purified sheep polyclonal antibody raised against the alphaC/alphaN subunit and an affinity-purified rabbit polyclonal antibody raised against the betaA subunit. The betaB subunit was below the level of detection in all human endometrial samples tested. Immunoreactive inhibin alphaC/alphaN subunit was localized in the luminal epithelium, glandular epithelium, stromal tissues and vascular endothelium with no significant variation across the normal menstrual cycle. Immunoreactive betaA subunit, common to inhibin A and activins AA and AB was localized in the luminal and glandular epithelium and in migratory cells while the endometrial stromal cells, decidua, vascular smooth muscle and endothelium were devoid of immunoreactivity. A significant variation of immunoreactive betaA subunit was observed in glandular and luminal epithelium across the normal menstrual cycle. In proliferative endometrium, only a very low level of betaA immunostaining was seen in luminal and glandular epithelium, while the luminal epithelial staining increased significantly in the early secretory phase and remained relatively constant over the rest of the menstrual cycle. A progressive increase in betaA immunoreactivity was observed also in the glandular epithelium during the secretory phase reaching a maximum in the late secretory phases, and decreasing at menstruation. Co-localization studies on serial sections suggested that the migratory cells expressing strong betaA immunoreactivity were macrophages and neutrophils but not eosinophils or mast cells. Thus, cells within the human endometrium are capable of expressing inhibin/activin molecules in vivo. The variation in the pattern of secretion of the betaA subunit across the menstrual cycle suggests that activin peptides may have a physiological role in endometrial function.     

Cloning, expression and characterisation of the cDNA encoding human hepatic squalene synthase, and its relationship to phytoene synthase

The reaction catalysed by squalene synthase (SQS) shows many similarities to that performed by another polyisoprene synthase, phytoene synthase (PhS). By identifying sequences conserved between yeast SQS (ySQS) and PhS, we have cloned a 2-kb cDNA (hSQS) encoding human SQS, a protein of 417 amino acids with a predicted M(r) of 48,041, which has only limited homology to ySQS. When expressed in E. coli, the hSQS cDNA directed the production of active enzyme. Two hSQS mRNA species of 2.0 and 1.55 kb have been identified which differ in their 3' untranslated sequences. The two mRNAs are present in roughly equal amounts in heart, placenta, lung, liver, kidney and pancreas, but the 2-kb mRNA predominates in brain and skeletal muscle. In HepG2 cells, both mRNAs are induced 2-4-fold by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin. In contrast, Northern blot analysis of rat tissues reveals only a 2.0-kb mRNA, which is considerably up-regulated in vivo by lovastatin.     

Elevated ovarian expression and serum concentration of alpha inhibin in the luteal phase during follicular development in the squirrel monkey (Saimiri boliviensis) compared to the human

The goal of the present investigation was to determine in the squirrel monkey the source and pattern of inhibin, a hormone known to effect reproductive steroid levels via pituitary and ovarian mechanisms. Since this seasonally polyestrous species is known to have elevated serum levels of reproductive steroids compared to other primates, the levels of ovarian alpha subunit mRNA expression and serum total alpha inhibin, estradiol, progesterone, and luteinizing hormone were measured and compared to human levels. Expression of the alpha subunit was robust in monkey luteal tissue compared to expression in human luteal tissue. Squirrel monkey serum inhibin peaked 4 days after the luteinizing hormone surge and correlated with progesterone changes. These luteal serum levels of inhibin were greater than 12 times higher than the human levels yet bio-LH activities were less than in the human during the luteal phase. Inhibin concentrations during the nonbreeding season were generally half the levels measured in the breeding season and undetectable in ovariectomized animals. However, exogenous FSH stimulation induced a marked rise in inhibin, which correlated with an estradiol rise. In conclusion, abundant alpha inhibin subunit expression in the luteal ovary of the squirrel monkey and loss of serum delectability in ovariectomized animals indicates that the principle source of inhibin in the squirrel monkey is the ovary. Elevated serum inhibin levels during the luteal phase concurrent with ovulatory-size follicular development is unique among species studied thus far. Possible simultaneous inhibin production from both follicular and luteal tissue may be responsible for the exceptionally high inhibin levels.